Antimicrobial peptides and methods of use thereof

ABSTRACT

Novel peptide analogs derived from the native sequences of CAP37 peptides 20-44 and 23-42, and their use as therapeutics against bacterial infections and diseases caused by bacterial infection. The peptide analog includes a serine or threonine substitution at one of the two cysteine residues at positions 26 and 42. Substitutions of the native peptide are also contemplated.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. Ser. No. 09/258,934, filed Mar. 1, 1999 nowU.S. Pat. No. 6,107,460.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Some aspect of this invention were made in the course of Grant RO1 AI28018 awarded by NIH, and therefore the government may have certainrights in some aspects of this invention.

BACKGROUND

The present invention is related to antimicrobial peptides and methodsof use thereof in vivo, and more particularly to substituted derivativesof peptide 20-44 of CAP37.

Antimicrobial therapies have advanced greatly over the years; however,people still die from infections and sepsis. The recent re-emergence ofold infections once thought to be on the decline and the rapid evolutionof resistant bacterial strains reinforces the critical need fordesigning and/or discovering new and more effective therapies. The mostsignificant development in it antibiotic therapy in the last decade hasbeen the exploitation of a group of naturally occurring host proteinsthat are potent antimicrobials, to produce more effective, safer andbroader acting drugs.

The immune system has at its disposal a number of mechanisms whereby itmay protect itself from invading pathogens. The bactericidal killingmechanisms of the human neutrophil are particularly effective andcomprise a collection of cationic granule proteins with potentantimicrobial action. One of these host-defense peptides is a novel,neutrophil protein known as CAP37 (Cationic Antimicrobial Protein ofMolecular weight 37 kDa). The first isolation and purification of CAP37was performed by Spitznagel and colleagues in 1984, who established thatCAP37 had very strong antimicrobial activity mainly againstgram-negative bacteria (1). Recently we demonstrated that in addition tothe previously demonstrated antibiotic activity, CAP37 is a potent andhighly specific chemoattractant for monocytes (2). We have determinedthe complete amino acid sequence of CAP37 (3) and cloned the gene forCAP37 (4). More recently we showed that CAP37 binds endotoxin (5).

Endotoxin is the outer membrane lipopolysaccharide (LPS) component ofgram-negative bacteria. Bacterial LPS has very important clinicalrelevance because of its pleiotropic effect on various immune cells. Itcan evoke various disease symptoms ranging from chills and fever tocirculatory collapse, multiorgan failure and death; a syndrome oftenreferred to as endotoxic or septic shock. Despite aggressive treatmentthe mortality rates remain high, with septic shock being the most commoncause of death in the intensive care unit and the thirteenth leadingcause of death overall. Figures from the Centers for Disease Control inAtlanta, Ga. suggest that septic shock occurs at the rate of 175 per100,000 people with the rate reaching almost 500 per 100,000hospitalized patients. The death rate is often as high as 25 to 40%.Septic/endotoxic shock has been shown to be most often due togram-negative bacteria although recent evidence would tend to indicatethat the incidence of shock due to gram-positive bacteria and fungi ison the rise. Factors that have contributed to the increasing incidenceof sepsis include the new immunosuppressive therapies, invasive devicessuch as intravenous catheters and surgical devices and an agingpopulation with many chronic diseases that predispose to sepsis.

The component responsible for the toxic effect of the LPS molecule isthe lipid component called lipid A. This region is embedded in the outermembrane of the bacterium and believed to be reasonably constant betweendifferent species of gram-negative bacteria. The manner in which LPSevokes its lethal effects is by binding to cells such as monocytes,macrophages and/or endothelial cells, triggering them to produce toxicoxygen radicals, cytokines such as tumor necrosis factor α (TNFα),various interleukins (IL-1, IL-6, and IL-8) and numerous other products.Our studies on CAP37 have led to the identification of a 25 amino acidpeptide that mimics the antimicrobial and lipopolysaccharide bindingfunctions of the native molecule (6). This synthetic peptide 20-44 basedon residues 20 through 44 of native CAP37 has the amino acid sequenceNQGRHFCGGALIHARFVMTAASCFQ (SEQ ID NO:1). This peptide has been tested invitro and in vivo for antimicrobial activity as well as endotoxinbinding activity (6). Peptide 20-44 shows strong in vitro bactericidalactivity mainly against gram-negative bacteria such as Salmonellatyphimurium, Escherichia coli and Pseudomonas aeruginosa. In addition,the peptide is active against the gram-positive organisms Enterococcusfaecalis and Staphylococcus aureus. In vivo experiments were conductedwith peptide 20-44 in a conscious rat model of sepsis using purifiedendotoxin (5). Intravenous infusion of peptide 20-44 (3.0 mg/kg bodyweight) with Escherichia coli LPS (250 μg/kg over 30 min) into consciousunrestrained rats prevented LPS-induced hyperdynamic and hypodynamiccirculatory shock. Peptide 20-44 (0.2, 1.0, and 5.0 mg/kg) administeredintravenously to conscious actinomycin-D sensitized rats following alethal dose of LPS neutralized LPS toxicity, resulting in dose-dependent7-day survival rates of 30, 50 and 80% respectively. Peptide 20-44 (5.0mg/kg) significantly inhibited the endotoxin-induced increase incirculating TNFαin sensitized rats. These data demonstrate that peptide20-44 has the capacity to abolish in vivo biological responses to LPSthat are relevant to human sepsis and to significantly neutralize thetoxicity of circulating Escherichia coli LPS.

The results described above, as well as further information regardingCAP37 peptides and their function are shown in U.S. Pat. Nos. 5,484,885;5,458,874; 5,607,916; 5,627,262; and 5,650,392, and pending U.S. Ser.No. 08/840,519, the specifications of all of which are herebyincorporated herein by reference in their entireties.

Table of Abbreviations - Amino Acids Name Abbreviations* Glycine Gly GAlanine Ala A Valine Val V Leucine Leu L Isoleucine Ile I Methionine MetM Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TCysteine Cys C Asparagine Asn N Glutamine Gln Q Tyrosine Tyr YTryptophan Trp W Aspartic acid Asp D Glutamic acid Glu E Histidine His HLysine Lys K Arginine Arg R *Where such abbreviations for amino acidsare used without an indication of enantiomeric structure, either the L-or D-enantiomers or a mixture of the L- or D-enantiomers may suitably beutilized.

BRIEF DESCRIPTION OF THE INVENTION

The present application describes new peptide analogs (e.g., see FIG. 1)based on the native sequence of CAP37 peptide 20-44 or 23-42 and theiruse as effective therapeutics against certain bacterial infections anddiseases caused by bacterial infection.

In a particularly preferred embodiment, the present inventioncontemplates a peptide, and a composition comprising said peptide, whichis a derivative of CAP37 peptide 23-42 (SEQ ID NO:2) wherein one of thecysteine residues at positions 26 and 42 is substituted with a serine orthreonine residue and one of the cysteine residues at positions 26 and42 is left unsubstituted. Further, the peptide derivative may compriseat least one of the following substitutions: phenylalanine replaced bytyrosine; glycine replaced by alanine; valine replaced by alanine,leucine, or isoleucine; alanine replaced by leucine, isoleucine orvaline; leucine replaced by alanine, isoleucine or valine; isoleucinereplaced by valine, leucine or alanine; serine replaced by histidine,arginine, or lysine; and threonine replaced by serine.

As noted elsewhere herein, the peptide derivative may be a derivative ofCAP 37 peptide 20-44 modified as described above for the derivative ofCAP37 peptide 23-42 above.

The invention further comprises a method of treating a bacterialinfection or septic shock in a patient, or prophylactically preventingseptic shock in a subject comprising administering a therapeuticallyeffective amount of a peptide derivative claimed and described herein.

Further, the peptide derivative contemplated herein may comprise apeptide (SEQ ID NO: 59 and SEQ ID NO: 60) having the formula:

R-H-X₁-X₂-X₃-X₄-X₅-X₆-X₇-H-X₈-R-X₉-X₁₀-M-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅

wherein X₁ and X₉ are phenylalanine and/or tyrosine; X₂ and X₁₅ arecysteine, serine, and/or threonine; X₃ and X₄ are glycine and/oralanine; X₅-X₈, X₁₀, X₁₂ and X₁₃ are alanine, leucine, isoleucine and/orvaline; X₁₁ is serine and/or threonine; X₁₄ is serine, threonine,histidine, arginine or lysine; R is arginine; H is histidine; M ismethionine; and with the proviso that one of X₂ and X₁₅ is cysteine andone of X₂ and X₁₅ is serine or threonine.

The present invention further comprises a DNA molecule having anucleotide sequence encoding a peptide having an amino acid sequence asdefined in any of the amino acid sequences listed or described herein,in particular, those having substituted cysteine residues at positions26 or 42.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a listing of the amino acid sequences of CAP37 peptide 20-44and analogs thereof.

FIG. 2 is a graph showing the bactericidal effects of peptides 20-44,20-44_(ser26) and 20-44_(ser42) at varying concentrations on Salmonellatyphimurium SH9178.

FIG. 3 is a graph showing the bactericidal effects of peptides 20-44,20-44_(ser26) and 20-44_(ser42) at varying concentrations on Salmonellatyphimurium LT2.

FIG. 4 is a graph showing the bactericidal effects of peptides 20-44,20-44_(ser26) and 20-44_(ser42) at varying concentrations onStaphylococcus aureus ATCC strain 25923.

FIG. 5 is a graph showing the bactericidal effects of peptides 20-44,20-44_(ser26) and 20-44_(ser42) at varying concentrations onEnterococcus faecalis.

FIG. 6 is a graph showing the binding of peptides 20-44_(ser26) to LipidA of LPS.

FIG. 7 is a graph showing the binding of peptide 20-44_(ser42) to LipidA of LPS.

FIG. 8 is a graph showing the effects of various peptides onLPS-mediated release of TNFα in rat macrophages.

FIG. 9 is a graph showing the effects of various concentrations ofpeptide 20-44 administered intravenously on survival of mice inoculatedwith live Salmonella typhimurium LT2.

FIG. 10 is a graph showing the effects of various concentrations ofpeptide 20-44 administered orally on survival of mice inoculated withlive Salmonella typhimurium LT2.

FIG. 11 is a graph showing the effects of various concentrations ofpeptide 20-44 administered intraperitoneally on survival of miceinoculated with live Salmonella typhimurium LT2.

FIG. 12 is a graph showing the effects of various concentrations ofpeptide 20-44_(ACM) administered intravenously on survival of miceinoculated with live Salmonella typhimurium LT2.

FIG. 13 is a graph showing the effects of various concentrations ofpeptide 20-44_(ACM) administered orally on survival of mice inoculatedwith live Salmonella typhimurium LT2.

FIG. 14 is a graph showing the effects of various concentrations ofpeptide 20-44_(ACM) administered intraperitoneally on survival of miceinoculated with live Salmonella typhimurium LT2.

FIG. 15 is a graph showing the effects of various concentrations ofpeptide 20-44_(ser26) administered intravenously on survival of miceinoculated with live Salmonella typhimurium LT2.

FIG. 16 is a graph showing the effects of various concentrations ofpeptide 20-44_(ser26) administered intraperitoneally on survival of miceinoculated with live Salmonella typhimurium LT2.

FIG. 17 is a graph showing the effects of various concentrations ofpeptide 20-44_(ser26) administered orally on survival of mice inoculatedwith live Salmonella typhimurium LT2.

FIG. 18 is a graph showing the effects of various concentrations ofpeptide 20-44_(ser42) administered intravenously on survival of miceinoculated with live Salmonella typhimurium LT2.

FIG. 19 is a graph showing the effects of various concentrations ofpeptide 20-44_(ser42) administered intraperitoneally on survival of miceinoculated with live Salmonella typhimuriumLT2.

FIG. 20 is a graph showing the effects of various concentrations ofpeptide 20-44_(ser42) administered orally on survival of mice inoculatedwith live Salmonella typhimurium LT2.

DETAILED DESCRIPTION OF THE INVENTION

The present application describes new peptide analogs (e.g., see FIG. 1)based on the native sequence of CAP37 peptide 20-44 or 23-42 and theiruse as effective therapeutics against certain bacterial infections anddiseases caused by bacterial infection.

In a particularly preferred embodiment, the present inventioncontemplates a peptide, and a composition comprising said peptide, whichis a derivative of CAP37 peptide 23-42 (SEQ ID NO:2) wherein one of thecysteine residues at positions 26 and 42 is substituted with a serine orthreonine residue and one of the cysteine residues at positions 26 and42 is left unsubstituted. Further, the peptide derivative may compriseat least one of the following substitutions: phenylalanine replaced bytyrosine; glycine replaced by alanine; valine replaced by alanine,leucine, or isoleucine; alanine replaced by leucine, isoleucine orvaline; leucine replaced by alanine, isoleucine or valine; isoleucinereplaced by valine, leucine or alanine; serine replaced by histidine,arginine, or lysine; and threonine replaced by serine.

As noted elsewhere herein, the peptide derivative may be a derivative ofCAP37 peptide 20-44 modified as described above for the derivative ofCAP37 peptide 23-42 above.

The invention further comprises a method of treating a bacterialinfection or septic shock in a patient, or prophylactically preventingseptic shock in a subject comprising administering a therapeuticallyeffective amount of a peptide derivative claimed and described herein.

Further, the peptide derivative contemplated herein may comprise apeptide (SEQ ID NO: 59 and SEQ ID NO: 60) having the formula:

R-H-X₁-X₂-X₃-X₄-X₅-X₆-X₇-H-X₈-R-X₉-X₁₀-M -X₁₁-X₁₂-X₁₃-X₁₄-X₁₅

wherein X₁ and X₉ are phenylalanine and/or tyrosine; X₂ and X₁₅ arecysteine, serine, and/or threonine; X₃ and X₄ are glycine and/oralanine; X₅-X₈, X₁₀, X₁₂ and X₁₃ are alanine, leucine, isoleucine and/orvaline; X¹¹ is serine and/or threonine; X₁₄ is serine, threonine,histidine, arginine or lysine; R is arginine; H is histidine; M ismethionine; and with the proviso that one of X₂ and X₁₅ is cysteine andone of X₂ and X₁₅ is serine or threonine.

The present invention further comprises a DNA molecule having anucleotide sequence encoding a peptide having an amino acid sequence asdefined in any of the amino acid sequences listed or described herein,in particular, those having substituted cysteine residues at positions26 or 42.

Peptide 20-44 of CAP37 in the native form contains two cysteine (cys)residues at positions 26 and 42 which together form a disulfide bridge(3). In order to assess the importance of these two cysteine residuesfor activity of the peptide, four analogs of the peptide weresynthesized and tested for their antibiotic action. Analogs20-44_(ser26) (SEQ ID NO:3), 20-44_(ser26) (SEQ ID NO:5), and20-44_(ser26/42) (SEQ ID NO:7) are homologous to the parent peptide,differing only in that serines were substituted for cysteines atpositions 26, 42, and 26 and 42, respectively. In the fourth peptide,20-44_(ACM), the sequence is the same as 20-44 (SEQ ID NO:1) except bothcysteines were permanently protected with an ACM(boc-cys-acetamidomethyl) side group and therefore could not readilyoxidize to disulfides (6).

Results described herein demonstrate that the novel peptides20-44_(ser 26) and 20-44_(ser 42) are effective in inhibiting growth ofcertain gram-negative and gram-positive bacteria and in blocking releaseof TNFα from LPS-stimulated macrophages. Methods used herein are thosepreviously described in U.S. Pats. No. 5,607,916; 5,627,262; and5,650,392.

The finding that derivatives of peptide 20-44 having a substitution ofonly a single cysteine residue are antimicrobial is a surprising result.Previous work indicated that both cysteines were necessary for activity.For example, U.S. Pat. No. 5,458,874 showed that a derivative of peptide20-44 having ACM side chains attached to each of the cysteine residuesat positions 26 and 42 (i.e., a peptide wherein disulfide bridgeformation was prevented), antimicrobial activity was substantiallyreduced (column 34, lines 41-52). When both cysteine residues atpositions 26 and 42 were replaced with serines, antimicrobial activitywas eliminated (U.S. Pat. No. 5,607,916, column 5, lines 6-10). It wasthus previously believed that the two cysteines at positions 26 and 42were necessary because of the requirement of a disulfide bridge, andformation of a peptide having a cyclic component (patent '916, column 6,lines 4-6).

These results would suggest that replacement of a single cysteineresidue would eliminate or significantly reduce antibacterial activity,contrary to what is taught, described, and enabled herein.

Antibiotic Peptides—in vitro Activity

Peptides 20-44, 20-44_(ser26) and 20-44_(ser42), were assayed forbactericidal activity using S. typhimurium SH9178 as the test organism.As shown in FIG. 2, peptide 20-44 had significant antibiotic activity.About 95% of the bacteria were killed at the highest concentration ofpeptide 20-44 tested (75 μM or 200 μg/ml), and 84% mortality wasachieved at 100 μg/ml of the peptide 20-44. Peptides 20-44_(ser26/42)and 20-44_(ACM) have been shown to be ineffective antibiotics (seecolumn 5, lines 6-10 in U.S. Pat. No. 5,607,916 and column 34, lines45-55 in U.S. Pat. No. 5,458,874). As described herein, peptides inwhich only one cys residue was replaced by serine (i.e. peptide20-44_(ser42) and peptide 20-44_(ser26)) were as active as the parentpeptide. Peptide 20-44_(ser42) killed 97.8% of bacteria at 75 μM, andpeptide 20-44_(ser26) killed 99.6% of bacteria at 75 μM. Importantly, atconcentrations of only 12.5 μM, peptide 20-44_(ser26) killed 70.4% oforganisms and peptide 20-44_(ser42) killed 66.6% of bacteria. We testedthese peptides with the smooth, more virulent LT2 strain of S.typhimurium, (FIG. 3). Both mono-substituted serine analogs showedstrong antibacterial activity against this organism as well. Indeed, thetwo serine analogs, 20-44_(ser26) and 20-44_(ser42) were able to kill73% and 65% of bacteria at 12.5 μM, the lowest concentration of peptidetested. These levels of mortality were very much greater than thoseobtained using peptide 20-44. Two other organisms, Staphylococcus aureusATCC strain 25923 and Enterococcus faecalis (a clinical isolate), bothof which are gram-positive organisms, were also tested against20-44_(ser26) and 20-44_(ser42). As indicated in FIGS. 4 and 5, bothmono-substituted serine analogs showed potent activity against these twobacterial strains.

LPS Binding Peptides—in vitro Sctivity

We have previously found that CAP37 binds lipopolysaccharide (LPS) (5,6) and that the LPS binding and antibacterial domain of CAP37 arecoincident (6). We tested the ability of the peptide analogs of thepresent invention to bind LPS in the in vitro limulus amebocyte lysate(LAL) assay and compared them with the parent peptide, 20-44. Analog20-44_(ser26) was capable of binding monophosphoryl lipid A, the toxiccomponent of the LPS molecule (FIG. 6). Similarly, peptide 20-44_(ser42)was also capable of neutralizing the effect of monophosphoryl lipid A atconcentrations greater than 500 μg/ml (FIG. 7) The effects of20-44_(ser26) at 500 μg/ml and at 1000 μg/ml were particularly potent,since it was able to reduce levels of free endotoxin (LPS) down tobaseline levels.

Effect of Peptides on LPS-mediated Release of TNFα

The effects of the serine-substituted peptides on LPS-mediated releaseof tumor necrosis factor α (TNFα) from rat peritoneal macrophages wasexplored. The deleterious effects of LPS in sepsis are due to itsinteraction with macrophages to produce a number of proinflammatorycytokines such as TNFα, interleukin-1 (IL-1), interleukin-6 (IL-6), andinterleukin- 8 (IL-8). Since peptide 20-44 and the twoserine-substituted analogs 20-44_(ser26) and 20-44_(ser42) bound LPS, wequestioned whether the peptides could bind LPS and neutralize the effectof LPS on macrophage release of cytokines. As indicated FIG. 8,20-44_(ser42) at 100 μg/ml was very effective at blocking TNFα releasefrom LPS stimulated macrophages. Levels of TNF-α were decreased by83.8%. Peptide 20-44_(Ser26) was also capable of neutralizing theeffects of LPS, as indicated by the reduction in TNFα levels by 42.9%.These are very significant findings since sometimes when septic patientsare treated with conventional antibiotics, they often become more ill.This is because LPS is shed from the bacterium as it is killed, and theconventional antibiotic cannot neutralize the effects of the free LPS.The fact that 20-44_(ser26) and 20-44_(ser42) can actually both killbacteria, and bind and neutralize LPS, make them very desirabletherapeutics for treating infections.

Antibiotic Peptides—in vivo Activity

Mice (female, Balb/c, 6-8 weeks) were administered a dose of live S.typhimurium LT2 (8×10⁸/mouse) via the oral route. Without treatment,this dose of live organisms led to the death of all animals between day3 and 4 (i.e. LD₁₀₀ at day 3 or 4). Following challenge with Salmonella,peptide 20-44 (0-150 μg/mouse) was administered via the intravenous,oral, and intraperitoneal routes. The results indicate that peptide20-44 when administered via the intravenous (FIG. 9), oral (FIG. 10),and intraperitoneal routes (FIG. 11), can rescue animals from lethalinfection due to S. typhimurium. At day 6 post infection, 100% ofanimals given a dose of 125 μg of peptide 20-44 intravenously survived.80% of mice given 50, 100 and 150 μg of peptide survived through to day5. Data shown in FIG. 10 strongly indicate that peptide 20-44 may alsobe given via the oral route since 80% survival rates were obtained with100 μg of peptide. This is evidence that the peptide is not destroyed bythe acid environment and enzymes of the stomach and indicates analternative route of administration to the intravenous route discussedin FIG. 9. The third route of administration of peptide 20-44 was theintraperitoneal route. This is often the method of choice in aveterinary situation. Importantly, the data in FIG. 11 show that thepeptide at a dose of 100 μg yields survival rates of 80% and 60% at days5 and 6 post infection, respectively. Mice treated with the controlpeptide, 20-44_(ACM), and vehicle/saline alone, succumbed to infectionvery rapidly after the inoculation with the bacteria (FIGS. 12-14).

On the other hand, peptide 20-44_(ser26) was highly effective atrescuing animals from lethal infection with Salmonella typhimurium. Atday 6 post infection, doses of peptide 20-44_(ser26) ranging from 25 to100 μg saved 80 to 100% of animals (FIG. 15). The efficacy of peptide20-44_(ser26) was also observed when administered via theintraperitoneal (FIG. 16) and oral routes (FIG. 17). Optimal doses ofpeptide given intraperitoneally rescued 80% of mice at day 6 postinfection and the oral administration of 75 μg of peptide 20-44_(ser26)saved 100% of animals at day 6. Untreated animals all succumbed toinfection between days 2 and 3. Survival rates with peptide20-44_(ser26) appeared to be greater than survival rates obtained withthe parent peptide.

Further, peptide 20-44_(ser42) appeared to be even more effective insaving animals from Salmonella infection than either the peptide 20-44or peptide 20-44_(ser26). Doses of 25 μg and 50 μg of peptide20-44_(SER42) given intravenously saved 100% of animals at 11 days postinfection (FIG. 18). The three doses of peptide 20-44_(ser42) givenintraperitoneally (FIG. 19) saved between 60% and 100% of mice infectedwith Salmonella. Oral doses of peptide 20-44_(ser42) between 100 and 125μg saved 80-100% of animals from infection (FIG. 20).

In summary, these data strongly support the notion that both analogs20-44_(ser26) and 20-44_(ser42) are potent antimicrobials. Anotherpeptide (20-44_(ACM)) in which both cysteines were blocked with theACM-side chain showed no activity. The two new analogs of peptide 20-44were also active against the gram-positive bacterial pathogens,Staphylococcus aureus and Enterococcus faecalis. Further, the datadescribed herein show that peptides 20-44_(ser26) and 20-44_(ser42) bindand neutralize the toxic effects of LPS endotoxin. Importantly, the invivo data using a live infection model in mice convincingly show thatthe peptides 20-44_(ser26) and 20-44_(ser42) can rescue mice from thelethal infection caused by Salmonella typhimurium. The efficacy of thenew peptides was demonstrated by their administration via theintravenous, intraperitoneal and oral routes.

In addition to the increased potency of peptide 20-44_(ser42) and20-44_(ser26), these two peptides have technical advantages over theparent peptide 20-44. The two new analogs are easier to synthesize andpurify because the internal disulfide bonding between the two cysteineresidues can no longer occur. The solubility of these two new peptidesis also greater than the 20-44 peptide, enabling their use at muchhigher concentrations than the 20-44 peptide.

The present invention further contemplates other analogs of peptide20-44 which have antimicrobial activity. For example, instead ofsubstituting either cysteine residue 26 or 42 with serine, they may besubstituted with threonine instead.

Further, without wishing to be constrained by theory, the antibioticactivity of peptide 20-44 is probably due to a combination of charge,hydrophobicity, ∝-helical structure, and the presence of cysteine. Thepresence of residues with basic groups is essential for microbicidalactivity. The entire native CAP37 molecule, for example, is stronglybasic. Peptide 20-44 has a charge of +2. However, at pH<6, the charge onthe peptide is +4 due to the presence of two histidines.

Since a combination of charge, hydrophobicity, and ∝-helix conformationis probably important for antimicrobial activity, the present inventioncontemplates that certain amino residues can be altered and/orsubstituted to enhance these features of the peptide. For example, thecharge of the peptide can be increased by replacing residues e.g., theserine residue at position 41 with arginine, histidine and/or lysineresidues as shown for example in FIG. 1, peptide analogs 20-44_(his41),20-44_(arg41) and 20-44_(lys41).

For example, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11 are the same asSEQ ID NO:1, except the serine residue at position 41 (position 22 inSEQ ID NO:1) has been replaced with histidine, arginine, and lysineresidues, respectively. SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 arethe same as SEQ ID NO:2, except the serine 41 residue has been replacedwith histidine, arginine, and lysine, respectively. SEQ ID NO:15, SEQ IDNO:16, and SEQ ID NO:17 are like SEQ ID NO:9, SEQ ID NO:10, and SEQ IDNO:11, respectively, except the cysteine at position 26 has beenreplaced by a serine. SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20 arelike SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively, exceptthe cysteine at position 42 has been replaced by a serine.

SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23 are like SEQ ID NO:12, SEQID NO:13, and SEQ ID NO:14, respectively, except the cysteine atposition 26 has been replaced with a serine. SEQ ID NO:24, SEQ ID NO:25,and SEQ ID NO:26 are like SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14,respectively, except the cysteine at position 42 has been replaced witha serine.

Replacement of amino acids (e.g. glycine) with low propensity for alphahelix formation with high propensity alanine residues would likelyincrease the effect of the alpha helicity, e.g., the replacement ofstrong helix breakers, gly 27 and gly 28 with alanine residues (see forexample peptide analogs 20-44_(ala27), 20-44_(ala28) and20-44_(ala27/28), of FIG. 1).

For example, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29 are like SEQID NO:1, except that (1) the second glycine (position 27) has beenreplaced with alanine, (2) the third glycine (position 28) has beenreplaced with alanine, and (3) both the second and third glycines(positions 27 and 28) have been replaced with alanines, respectively(see FIG. 1: 20-44_(ala27), 20-44_(ala28) and 20-44_(ala27/28)). Each ofthese sequences may be further modified by replacing either cysteineresidue. For example, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32 arelike SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, respectively, exceptthe cysteine₂₆ has been replaced with serine. SEQ ID NO:33, SEQ IDNO:34, and SEQ ID NO:35 are like SEQ ID NO:27, SEQ ID NO:28, and SEQ IDNO:29, respectively, except the cysteine₄₂ has been replaced withserine.

It is further contemplated where used herein, threonine may be used as asubstitute for cysteine in lieu of a serine residue (see for examplepeptide 20-44_(thr) on FIG. 1 and SEQ ID NO:36). In other words, anyanalog described herein in which a cysteine has been substituted with aserine, the cysteine could instead be substituted with a threonine toobtain an analog having a similar activity.

Moreover, it is further contemplated that the valine at position 36could be substituted with a leucine, isoleucine, or alanine residue andstill provide an analog which maintained antimicrobial activity ascontemplated herein (see analogs 20-44_(leu36), 20-44_(ile36), and20-44_(ala36) on FIG. 1 and SEQ ID NO:37, SEQ ID NO:38, and SEQ IDNO:39). Further, it is contemplated that one, two, or all three of thephenylalanine residues at positions 25, 35, and 43 could be substitutedwith a tyrosine residue (see analogs 20-44_(tyr25), 20-44_(tvr35) and20-44_(tyr43) of FIG. 1 and SEQ ID NO:40, SEQ ID NO:41, and SEQ IDNO:42.)

It is further contemplated that alanine residues in the peptide could bereplaced with valine, leucine, or isoleucine. Similarly, leucineresidues could be replaced with alanine, valine, or isoleucine.Similarly, isoleucine could be replaced with alanine, valine, or leucineresidues.

It is contemplated that any of the substitutions described herein forpeptide 20-44 can also be made for corresponding residues of peptide23-42.

Peptide 23-42_(ser26) may for example be substituted with alanine atposition 27 or 28 or at both positions 27 and 28, leucine, isoleucine,or alanine at position 36, or tyrosine at positions 25, or 35, as seenin SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50.

Peptide 23-42_(ser42) may be substituted with alanine at position 27 or28 or at both positions 27 and 28, leucine, isoleucine or alanine atposition 36, or tyrosine at positions 25, or 35, as seen in SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, and SEQ ID NO:58. Each of SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56,SEQ ID NO:57, and SEQ ID NO:58, may instead have a threonine residueused in lieu of the serine residue at positions 26 or 42.

Utility

The present invention contemplates using the novel peptides describedherein and/or effective subunits thereof both to treat ongoing endotoxic(septic) shock or bacterial infection and to prophylactically treat anindividual who may have a risk of septic shock prior to a surgicalprocedure such as bowel or bladder surgery or surgical manipulation ofother organs where gram-negative bacteria normally reside and couldenter the bloodstream.

The peptide, synthetically or recombinantly produced, may be used as apharmaceutical composition when combined with a pharmaceuticallyacceptable carrier. Such a composition may contain, in addition to thepeptide and carrier, diluents, fillers, salts, buffers, stabilizers,solubilizers, and other materials well known in the art. Suitablecarriers, vehicles and other components of the formulation aredescribed, for example, in Remingtons' Pharmaceutical Sciences, (MackPublishing Co., 1980). The term “pharmaceutically acceptable” means anon-toxic material that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). The characteristics ofthe carrier will depend on the route of administration.

The pharmaceutical composition of the invention may be in the form of aliposome in which isolated peptide is combined, in addition to otherpharmaceutically acceptable carriers, with amphipathic agents such aslipids which exist in aggregated form as micelles, insoluble monolayers,liquid crystals, or lamellar layers which in aqueous solution. Suitablelipids for liposomal formulation include, without limitation,monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,saponin, bile acids, and the like. Preparation of such liposomalformulations is within the level of skill in the art, as disclosed, forexample, in U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat.No. 4,837,028; and U.S. Pat. No. 4,737,323, all of which areincorporated herein by reference.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, i.e.,reduction of infection or sepsis. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the therapeutic effect,whether administered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of the peptide composition isadministered to a mammal having a bacterial disease state. Peptide maybe administered in accordance with the method of the invention eitheralone or in combination with other therapies.

Administration of peptide used in the pharmaceutical composition or topractice the method of the present invention can be carried out in avariety of conventional ways, such as oral ingestion, inhalation, orcutaneous, subcutaneous, intraperitoneal, or intravenous injection.

When a therapeutically effective amount of peptide is administeredorally, the peptide will be in the form of a tablet, capsule, powder,solution or elixir. When administered in tablet form, the pharmaceuticalcomposition of the invention may additionally contain a solid carriersuch as a gelatin or an adjuvant. The tablet, capsule, and powderpreferably contains from about 5 to 95% peptide. When administered inliquid form, a liquid carrier such as water, petroleum, oils of animalor plant origin such as peanut oil, mineral oil, soybean oil, or sesameoil, or synthetic oils may be added. The liquid form of thepharmaceutical composition may further contain physiological salinesolution, dextrose or other saccharide solution, or glycols such asethylene glycol, 35 propylene glycol or polyethylene glycol. Whenadministered in liquid form, the pharmaceutical composition preferablycontains from about 0.5 to 90% by weight of peptide.

When a therapeutically effective amount of peptide is administered byintravenous, cutaneous or subcutaneous injection, peptide will be in theform of a pyrogen-free, parenterally acceptable aqueous solution. Thepreparation of such parenterally acceptable peptide solutions, havingdue regard to pH, isotonicity, stability, and the like, is within theskill in the art. A preferred pharmaceutical composition forintravenous, cutaneous, or subcutaneous injection should contain, inaddition to peptide an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additive known to those of skill in the art.

The invention further includes a method of treating a wound by topicalapplication of a composition containing one or more peptides as definedherein which possess antibacterial activity in a pharmacologicallyeffective amount to promote wound healing and/or treat infection. Otheradditions to the medication may be desirable such as the inclusion ofepidermal growth factor also present in a pharmacologically effectiveamount to promote wound healing. The topical medication may take anynumber of standard forms such as pastes, gels, creams, and ointments.

The amount of peptide in the pharmaceutical composition of the presentinvention will depend upon the nature and severity of the conditionbeing treated, and on the nature of prior treatments which the patienthas undergone. Ultimately, the attending physician will decide theamount of peptide with which to treat each individual patient.Initially, the attending physician will preferably administer low dosesof peptide and observe the patient's response. Larger doses of peptidemay be administered until the optimal therapeutic effect is obtained forthe patient, and at that point the dosage is not increased further.Without wishing to be held to a specific dosage, it is contemplated thatthe various pharmaceutical compositions used to practice the method ofthe present invention should contain about 0.1 μg to about 100 mg ofpeptide per kg body weight.

The duration of intravenous therapy using the pharmaceutical compositionof the present invention will vary, depending on the severity of thedisease being treated and the condition and potential idiosyncraticresponse of each individual patient. It is contemplated that theduration of each application of the peptide will be in the range of 1 to12 to 24 hours of continuous intravenous administration. Ultimately theattending physician will decide on the appropriate duration ofintravenous therapy using the pharmaceutical composition of the presentinvention.

Other antibiotics, intravenous fluids, cardiovascular and respiratorysupport could also be provided if requested by the attending physicianin a manner known to one of ordinary skill in the art.

Endotoxin contamination of research reagents including aqueous buffers,cell culture media, pharmacological agents, solutions containingbioactive proteins and mediators are a common problem often leading toserious artifacts thereby confounding many experimental outcomes.

The peptide derivatives based on the CAP37 sequence 20-44 as describedherein afford a convenient and efficient method for detoxifying orremoving contaminating endotoxin from an aqueous sample using affinitychromatography techniques. The peptide is coupled to an inert supportand a chromatographic column is prepared. Because of the strong affinityof CAP37 peptides for endotoxin, any endotoxin present in a sampleapplied to the column, will bind to the peptide on the support. Thesample that is eluted or passes through the column will therefore bedevoid of endotoxin.

Affinity chromatography is a widely used procedure for the purificationof proteins (11). In general the procedure consists of coupling thepeptide to a suitable matrix, such as agarose or cellulose. The matrixmaterial is chosen such that it is hydrophilic, since this reducesnon-specific interactions. It must also have large pores, and be rigidso as to be able to resist packing and washing with various buffers. Itmust also be chemically inert and stable to enable as wide a spectrum ofsolvents to be applied during the separation (11).

The coupling of peptide to matrix is according to standard procedureswell known to those of ordinary skill in the art and can involve the useof a number of linking groups including cyanogen bromide, tresyl, epoxyand triazine. Coupling is performed under controlled conditions of pH,ionic strength and ion content at room temperature or 4° C. for 4-16hours. Excess ligand is removed and the unreacted sites on the matrixare blocked to diminish non-specific interaction. The ligand-coupledmatrix can be used to separate endotoxin contaminated samples eitherthrough batch absorption or by using it packed in a column. For eitherformat the appropriate buffers need to be used. The best bufferconditions utilize either phosphate or Tris buffers (0.1-0.2 M)containing salts (NaCl, 0.1-0.5M). For the column format, thematrix-ligand slurry is loaded into a column of suitable dimensions toaccommodate the sample volume. The gel is allowed to settle and thesample is loaded. The elution is performed using the buffer conditionslisted, and the sample minus endotoxin that passes through the columncollected in sterile -pyrogen-free containers.

Therefore, the invention as contemplated herein further comprises anaffinity chromatography column or support matrix comprising a peptide asdescribed herein.

All references cited herein are hereby incorporated by reference herein.

The present invention is not to be limited in scope by the specificembodiments described herein, since such embodiments are intended as butsingle illustrations of one aspect of the invention and any functionallyequivalent embodiments are within the scope of this invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

References

1. Shafer, W. M., L. E. Martin and J. K. Spitznagel 1984 Cationicantimicrobial proteins isolated from human neutrophil granulocytes inthe presence of diisopropyl fluorophosphate. Infect. Immun. 45:29-35.

2. Pereira, H. A., W. M. Shafer, J. Pohl, L. E. Martin, and J. K.Spitznagel. 1990. CAP37, a human neutrophil-derived chemotactic factorwith monocyte specific activity. J. Clin. Invest. 85: 1468-1476

3. Pohl, J., H. A. Pereira, N. M. Martin, and J. K. Spitznagel 1990.Amino acid sequence of CAP37, a human neutrophil granule-derivedantibacterial and monocyte-specific chemotactic glycoproteinstructurally similar to neutrophil elastase. FEBS Lett. 272: 200-204.

4. Morgan, J. G., T. Sukiennicki, H. A. Pereira, J. K. Spitznagel, M. E.Guerra, and J. W. Larrick. 1991. Cloning of cDNA for the serine proteasehomolog CAP37/azurocidin, a microbicidal and chemotactic protein fromhuman granulocytes. J. Immunol. 147: 3210-3214.

5. Brackett, D. J., M. R. Lerner, M. A. Lacquement, R. He, and H. A.Pereira. 1997. A synthetic lipopolysaccharide-binding peptide based onthe neutrophil-derived protein CAP37 prevents endotoxin-inducedresponses in conscious rats. Infect. Immun. 65: 2803-2811

6. Pereira, H. A., I. Erdem, J. Pohl, and J. K. Spitznagel. 1993.Synthetic bactericidal peptide based on CAP37: A 37 kDa human neutrophilgranule-associated cationic antimicrobial protein chemotactic formonocytes. Proc. Natl. Acad. Sci. (USA). 90:4733-4737

7. Fink, J., A. Boman, H. G. Boman, and R. B. Merrifield. 1989. Design,synthesis and antibacterial activity of cecropin-like model peptides.Int. J. Peptide Protein Res. 33: 412-421.

8. Andreu, D., R. B. Merrifield, H. Steiner, and H. G. Boman. 1985N-terminal analogues of cecropin A: synthesis, antibacterial activity,and conformational properties. Biochemistry 24: 1683-1688.

9. Raj, P. A., M. Edgerton, and M. J. Levine. 1990. Salivary histatin 5.Dependence of sequence, chain length, and helical conformation forcandidacidal activity. J. Biol. Chem. 265: 3898-3905

10. Berkowitz, B. A., C. L. Bevins, and M. A. Zasloff. 1990. Magainins:a new family of membrane active host defense peptides. Biochem.Pharmacol. 39: 65-629.

11. Ostrove, S. Affinity Chromatography: General Methods. In, Methods inEnzymology, 182: 357-375. Ed. M. P. Deutscher, Academic Press, San Diego1990.

60 1 25 PRT Homo Sapiens 1 Asn Gln Gly Arg His Phe Cys Gly Gly Ala LeuIle His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Ser Cys Phe Gln 20 252 20 PRT Homo Sapiens 2 Arg His Phe Cys Gly Gly Ala Leu Ile His Ala ArgPhe Val Met Thr 1 5 10 15 Ala Ala Ser Cys 20 3 25 PRT Homo Sapiens 3 AsnGln Gly Arg His Phe Ser Gly Gly Ala Leu Ile His Ala Arg Phe 1 5 10 15Val Met Thr Ala Ala Ser Cys Phe Gln 20 25 4 20 PRT Homo Sapiens 4 ArgHis Phe Ser Gly Gly Ala Leu Ile His Ala Arg Phe Val Met Thr 1 5 10 15Ala Ala Ser Cys 20 5 25 PRT Homo Sapiens 5 Asn Gln Gly Arg His Phe CysGly Gly Ala Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala SerSer Phe Gln 20 25 6 20 PRT Homo Sapiens 6 Arg His Phe Cys Gly Gly AlaLeu Ile His Ala Arg Phe Val Met Thr 1 5 10 15 Ala Ala Ser Ser 20 7 25PRT Homo Sapiens 7 Asn Gln Gly Arg His Phe Ser Gly Gly Ala Leu Ile HisAla Arg Phe 1 5 10 15 Val Met Thr Ala Ala Ser Ser Phe Gln 20 25 8 20 PRTHomo Sapiens 8 Arg His Phe Ser Gly Gly Ala Leu Ile His Ala Arg Phe ValMet Thr 1 5 10 15 Ala Ala Ser Ser 20 9 20 PRT Homo Sapiens 9 Arg His PheSer Gly Gly Ala Leu Ile His Ala Arg Phe Val Met Thr 1 5 10 15 Ala AlaSer Ser 20 10 25 PRT Homo Sapiens 10 Asn Gln Gly Arg His Phe Cys Gly GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Arg Cys PheGln 20 25 11 25 PRT Homo Sapiens 11 Asn Gln Gly Arg His Phe Cys Gly GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Lys Cys PheGln 20 25 12 20 PRT Homo Sapiens 12 Arg His Phe Cys Gly Gly Ala Leu IleHis Ala Arg Phe Val Met Thr 1 5 10 15 Ala Ala His Cys 20 13 20 PRT HomoSapiens 13 Arg His Phe Cys Gly Gly Ala Leu Ile His Ala Arg Phe Val MetThr 1 5 10 15 Ala Ala Arg Cys 20 14 20 PRT Homo Sapiens 14 Arg His PheCys Gly Gly Ala Leu Ile His Ala Arg Phe Val Met Thr 1 5 10 15 Ala AlaLys Cys 20 15 25 PRT Homo Sapiens 15 Asn Gln Gly Arg His Phe Ser Gly GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala His Cys PheGln 20 25 16 25 PRT Homo Sapiens 16 Asn Gln Gly Arg His Phe Ser Gly GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Arg Cys PheGln 20 25 17 25 PRT Homo Sapiens 17 Asn Gln Gly Arg His Phe Ser Gly GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Lys Cys PheGln 20 25 18 25 PRT Homo Sapiens 18 Asn Gln Gly Arg His Phe Cys Gly GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala His Ser PheGln 20 25 19 25 PRT Homo Sapiens 19 Asn Gln Gly Arg His Phe Cys Gly GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Arg Ser PheGln 20 25 20 25 PRT Homo Sapiens 20 Asn Gln Gly Arg His Phe Cys Gly GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Lys Ser PheGln 20 25 21 20 PRT Homo Sapiens 21 Arg His Phe Ser Gly Gly Ala Leu IleHis Ala Arg Phe Val Met Thr 1 5 10 15 Ala Ala His Cys 20 22 20 PRT HomoSapiens 22 Arg His Phe Ser Gly Gly Ala Leu Ile His Ala Arg Phe Val MetThr 1 5 10 15 Ala Ala Arg Cys 20 23 20 PRT Homo Sapiens 23 Arg His PheSer Gly Gly Ala Leu Ile His Ala Arg Phe Val Met Thr 1 5 10 15 Ala AlaLys Cys 20 24 20 PRT Homo Sapiens 24 Arg His Phe Cys Gly Gly Ala Leu IleHis Ala Arg Phe Val Met Thr 1 5 10 15 Ala Ala His Ser 20 25 20 PRT HomoSapiens 25 Arg His Phe Cys Gly Gly Ala Leu Ile His Ala Arg Phe Val MetThr 1 5 10 15 Ala Ala Arg Ser 20 26 20 PRT Homo Sapiens 26 Arg His PheCys Gly Gly Ala Leu Ile His Ala Arg Phe Val Met Thr 1 5 10 15 Ala AlaLys Ser 20 27 25 PRT Homo Sapiens 27 Asn Gln Gly Arg His Phe Cys Ala GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Ser Cys PheGln 20 25 28 25 PRT Homo Sapiens 28 Asn Gln Gly Arg His Phe Cys Gly AlaAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Ser Cys PheGln 20 25 29 25 PRT Homo Sapiens 29 Asn Gln Gly Arg His Phe Cys Ala AlaAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Ser Cys PheGln 20 25 30 25 PRT Homo Sapiens 30 Asn Gln Gly Arg His Phe Ser Ala GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Ser Cys PheGln 20 25 31 25 PRT Homo Sapiens 31 Asn Gln Gly Arg His Phe Ser Gly AlaAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Ser Cys PheGln 20 25 32 25 PRT Homo Sapiens 32 Asn Gln Gly Arg His Phe Ser Ala AlaAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Ser Cys PheGln 20 25 33 25 PRT Homo Sapiens 33 Asn Gln Gly Arg His Phe Cys Ala GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Ser Ser PheGln 20 25 34 25 PRT Homo Sapiens 34 Asn Gln Gly Arg His Phe Cys Gly AlaAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Ser Ser PheGln 20 25 35 25 PRT Homo Sapiens 35 Asn Gln Gly Arg His Phe Cys Ala AlaAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Ser Ser PheGln 20 25 36 25 PRT Homo Sapiens 36 Asn Gln Gly Arg His Phe Cys Gly GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Thr Cys PheGln 20 25 37 25 PRT Homo Sapiens 37 Asn Gln Gly Arg His Phe Cys Gly GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Leu Met Thr Ala Ala Ser Cys PheGln 20 25 38 25 PRT Homo Sapiens 38 Asn Gln Gly Arg His Phe Cys Gly GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Ile Met Thr Ala Ala Ser Cys PheGln 20 25 39 25 PRT Homo Sapiens 39 Asn Gln Gly Arg His Phe Cys Gly GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Ala Met Thr Ala Ala Ser Cys PheGln 20 25 40 25 PRT Homo Sapiens 40 Asn Gln Gly Arg His Tyr Cys Gly GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Ser Cys PheGln 20 25 41 25 PRT Homo Sapiens 41 Asn Gln Gly Arg His Phe Cys Gly GlyAla Leu Ile His Ala Arg Tyr 1 5 10 15 Val Met Thr Ala Ala Ser Cys PheGln 20 25 42 25 PRT Homo Sapiens 42 Asn Gln Gly Arg His Phe Cys Gly GlyAla Leu Ile His Ala Arg Phe 1 5 10 15 Val Met Thr Ala Ala Ser Cys TyrGln 20 25 43 20 PRT Homo Sapiens 43 Arg His Phe Ser Ala Gly Ala Leu IleHis Ala Arg Phe Val Met Thr 1 5 10 15 Ala Ala Ser Cys 20 44 20 PRT HomoSapiens 44 Arg His Phe Ser Gly Ala Ala Leu Ile His Ala Arg Phe Val MetThr 1 5 10 15 Ala Ala Ser Cys 20 45 20 PRT Homo Sapiens 45 Arg His PheSer Ala Ala Ala Leu Ile His Ala Arg Phe Val Met Thr 1 5 10 15 Ala AlaSer Cys 20 46 20 PRT Homo Sapiens 46 Arg His Phe Ser Gly Gly Ala Leu IleHis Ala Arg Phe Leu Met Thr 1 5 10 15 Ala Ala Ser Cys 20 47 20 PRT HomoSapiens 47 Arg His Phe Ser Gly Gly Ala Leu Ile His Ala Arg Phe Ile MetThr 1 5 10 15 Ala Ala Ser Cys 20 48 20 PRT Homo Sapiens 48 Arg His PheSer Gly Gly Ala Leu Ile His Ala Arg Phe Ala Met Thr 1 5 10 15 Ala AlaSer Cys 20 49 20 PRT Homo Sapiens 49 Arg His Tyr Ser Gly Gly Ala Leu IleHis Ala Arg Phe Val Met Thr 1 5 10 15 Ala Ala Ser Cys 20 50 20 PRT HomoSapiens 50 Arg His Phe Ser Gly Gly Ala Leu Ile His Ala Arg Tyr Val MetThr 1 5 10 15 Ala Ala Ser Cys 20 51 20 PRT Homo Sapiens 51 Arg His PheCys Ala Gly Ala Leu Ile His Ala Arg Phe Val Met Thr 1 5 10 15 Ala AlaSer Ser 20 52 20 PRT Homo Sapiens 52 Arg His Phe Cys Gly Ala Ala Leu IleHis Ala Arg Phe Val Met Thr 1 5 10 15 Ala Ala Ser Ser 20 53 20 PRT HomoSapiens 53 Arg His Phe Cys Ala Ala Ala Leu Ile His Ala Arg Phe Val MetThr 1 5 10 15 Ala Ala Ser Ser 20 54 20 PRT Homo Sapiens 54 Arg His PheCys Gly Gly Ala Leu Ile His Ala Arg Phe Leu Met Thr 1 5 10 15 Ala AlaSer Ser 20 55 20 PRT Homo Sapiens 55 Arg His Phe Cys Gly Gly Ala Leu IleHis Ala Arg Phe Ile Met Thr 1 5 10 15 Ala Ala Ser Ser 20 56 20 PRT HomoSapiens 56 Arg His Phe Cys Gly Gly Ala Leu Ile His Ala Arg Phe Ala MetThr 1 5 10 15 Ala Ala Ser Ser 20 57 20 PRT Homo Sapiens 57 Arg His TyrCys Gly Gly Ala Leu Ile His Ala Arg Phe Val Met Thr 1 5 10 15 Ala AlaSer Ser 20 58 20 PRT Homo Sapiens 58 Arg His Phe Cys Gly Gly Ala Leu IleHis Ala Arg Tyr Val Met Thr 1 5 10 15 Ala Ala Ser Ser 20 59 20 PRTArtificial sequence Completely synthesized 59 Arg His Xaa Cys Xaa XaaXaa Xaa Xaa His Xaa Arg Xaa Xaa Met Xaa 1 5 10 15 Xaa Xaa Xaa Xaa 20 6020 PRT Artificial sequence Completely synthesized 60 Arg His Xaa Xaa XaaXaa Xaa Xaa Xaa His Xaa Arg Xaa Xaa Met Xaa 1 5 10 15 Xaa Xaa Xaa Cys 20

What is claimed is:
 1. A DNA molecule comprising a nucleotide sequenceencoding a peptide having the amino acid sequence:R-H-X₁-X₂-X₃-X₄-X₅-X₆-X₇-H-X8-R-X₉-X₁₀-M-X₁₁-X₂-X₁₃--X₁₄-X₁₅ wherein: X₁is phe or tyr; X₂ is cys, ser, or thr; X₃ is gly or ala; X₄ is gly orala; X₅-X₈, X₁₀, X₁₂ and X₁₃ are ala, leu, ile or val; X₉ is phe or tyr;X₁₁ is ser or thr; X₁₄ is ser, thr, his, arg or lys; X₁₅ is ser, cys orthr; R is arg; H is his; and M is met; and with the proviso that when X₂is cys, X₁₅ is ser or thr and when X₁₅ is cys, X₂ is ser or thr.